This application claims priority to Ser. No. 60/711,451 filed Aug. 25, 2005, the entire contents of which are incorporated herein by reference. Without limiting the scope of the invention, its background is described in connection with antiviral agents.
Viral diseases have remained a major global health threat. It would have been a far more devastating epidemic if there were no innate immunity, which is the first line of defense against microbial pathogens. How viruses elicit and evade host innate immune defense is not well understood.
Viral infection of host cells triggers innate and adaptive immune responses that are essential for the survival of the host. A highly effective antiviral immune response is the production of type-I interferons, such as interferon-α (IFN-α) and interferon-β (IFN-β). These interferons activate the JAK-STAT pathway to stimulate the expression of interferon-stimulated genes (ISGs), which collectively inhibit viral replication and assembly (Darnell et al., 1994). The genes encoding interferons are regulated by the assembly of an enhanceosome containing several transcription factors including NF-κB and IRF3, both of which are regulated by subcellular localization (Maniatis et al., 1998). In unstimulated cells, NF-κB is sequestered in the cytoplasm in association with an inhibitor of the IκB family (Baeuerle and Baltimore, 1988). Stimulation of cells with cytokines (e.g., TNFα or IL-1β) or pathogens (e.g., bacteria or viruses) leads to the activation of a large kinase complex consisting of the catalytic subunits IKKα and IKKβ, and the essential regulatory subunit NEMO (also known as IKKγ or IKKAP). The activated IKK complex phosphorylates IκB and targets this inhibitor for degradation by the ubiquitin-proteasome pathway. NF-κB is then liberated to enter the nucleus to turn on a battery of genes essential for immune and inflammatory responses (Silverman and Maniatis, 2001).
Similar to NF-κB, IRF3 is also retained in the cytoplasm of unstimulated cells. After viral or bacterial infection of cells, IRF3 is phosphorylated at multiple serine and threonine residues at the C-terminus (Hiscott et al., 2003; Yoneyama et al., 2002). The phosphorylated IRF3 then homodimerizes and enters the nucleus to activate IFN-β in a highly cooperative manner with NF-κB. The kinases that phosphorylate IRF3 have recently been identified as the IKK-like kinases TBK1 and IKKε (Fitzgerald et al., 2003; Sharma et al., 2003). Genetic studies have shown that TBK1 is important for interferon production by the bacterial cell wall component lipopolysaccharides (LPS) as well as viruses (Hemmi et al., 2004; McWhirter et al., 2004). In some cells, however, the function of TBK1 may be compensated by IKKε or other kinases (Perry et al., 2004; Yoneyama et al., 2004). TBK1 and IKKε can also phosphorylate and activate IRF7 (tenOever et al., 2004), another IRF family member essential for the production of type-I interferons such as interferon-α (Honda et al., 2005).
Both NF-κB and IRFs are tightly regulated by microbial pathogens, including RNA viruses. After entry into host cell and uncoating of an RNA virus, the viral RNA replicates to produce double-stranded RNA intermediates, which are recognized by the host as a pathogen-associated molecular pattern (PAMP). Several proteins that recognize viral RNA have been discovered, including Toll-like receptors TLR3, 7 and 8 (Akira and Takeda, 2004). TLR3 contains an intracellular Toll-Interleukin Receptor (TIR) domain that signals to NF-κB and IRF3 via the adaptor protein TRIF (Akira and Takeda, 2004), whereas the TIR domain of TLR7 and TLR8 binds to another adaptor MyD88, which associates with and activates IRF7 to induce interferon-α (Kawai et al., 2004). In addition, TLRs contain several extracellular Leucine-Rich Repeats (LRR) that presumably recognize microbial ligands. Thus, the topology of these receptors dictates that they can only recognize extracellular dsRNA or single-stranded RNA associated with viral particles that are internalized into the endosomes (Crozat and Beutler, 2004). The receptor that detects intracellular dsRNA generated by the virus is a newly identified protein RIG-I, which has two N-terminal caspase activation and recruitment domains-like (CARD-like) domains, and a C-terminal RNA helicase domain that binds to dsRNA (Sumpter et al., 2005; Yoneyama et al., 2004). Presumably, the binding of viral RNA to RIG-I leads to a conformational change that exposes the CARD-like domain, which then activates downstream signaling. Consistent with this model, overexpression of the N-terminal CARD-like domains of RIG-I is sufficient to activate both NF-κB and IRF3 (Yoneyama et. al., 2004). However, the mechanism by which RIG-I activates NF-κB and IRF3. is currently not understood. A RIG-I-like protein MDA-5 (also known as HELICARD), which also contains two CARD-like domains and an RNA helicase domain, has recently been shown to be involved in dsRNA signaling and apoptosis (Andrejeva et al., 2004; Kang et al., 2002; Kovacsovics et al., 2002). However, MDA-5 does not appear to function redundantly with RIG-I in the antiviral pathway, as an inactivating mutation of RIG-I or the loss of RIG-I expression completely blocks interferon production by several RNA viruses (Kato et al., 2005; Sumpter et al., 2005; Yoneyama et al., 2004).